WO2021136249A1 - Anticorps bispécifique induit par une modification de fab, son procédé de préparation et son utilisation - Google Patents

Anticorps bispécifique induit par une modification de fab, son procédé de préparation et son utilisation Download PDF

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WO2021136249A1
WO2021136249A1 PCT/CN2020/140730 CN2020140730W WO2021136249A1 WO 2021136249 A1 WO2021136249 A1 WO 2021136249A1 CN 2020140730 W CN2020140730 W CN 2020140730W WO 2021136249 A1 WO2021136249 A1 WO 2021136249A1
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heavy chain
light chain
mutated
charged amino
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周易
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周易
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/46—Hybrid immunoglobulins
    • C07K16/468—Immunoglobulins having two or more different antigen binding sites, e.g. multifunctional antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2863—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against receptors for growth factors, growth regulators
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/32—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against translation products of oncogenes
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/40—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against enzymes
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/46—Hybrid immunoglobulins
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/10—Cells modified by introduction of foreign genetic material
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/31—Immunoglobulins specific features characterized by aspects of specificity or valency multispecific
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
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    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value

Definitions

  • the invention belongs to the field of antibody engineering, and specifically relates to a bispecific antibody induced by Fab modification and a preparation method and application thereof.
  • IgG type bispecific antibodies have similar structure, physicochemical properties and Fc segment functions to common antibodies.
  • an IgG type bispecific antibody consists of two heavy chains with different amino acid sequences (i.e. heavy chain HC_A against antigen A and heavy chain HC_B against antigen B) and two light chains with different amino acid sequences (i.e. light chain against antigen A).
  • the chain LC_A and the anti-antigen B light chain LC_B) are composed.
  • 4 polypeptide chains are combined, homodimers and heterodimers will be formed between the two heavy chains, and mismatches will also be formed between the light and heavy chains. Therefore, there will be 8 different combinations, of which only One is the desired target antibody molecule. However, it is extremely inefficient and difficult to separate and purify the target molecule from 8 kinds of molecules.
  • IgG-type bispecific antibodies promote the formation of heterodimers between the two heavy chains of the antibody by modifying the Fc segment (Ridgway, Presta et al. 1996; Carter 2001, US2010286374A1, CN106883297A, US20150307628A1).
  • the ideal state is that of the two heavy chains and the two light chains that constitute the bispecific antibody, the light chain LC_A only specifically pairs with the heavy chain HC_A, but not with the heavy chain HC_B.
  • Chain LC_B only specifically paired with heavy chain HC_B, but not with heavy chain HC_A.
  • the purpose of the present invention is to overcome the deficiencies in the prior art and provide a bispecific antibody induced by Fab transformation and its preparation method and application.
  • amino acid modifications are introduced at specific positions on the antibody light-heavy chain interaction interface, and connecting peptides are introduced into the heavy and light chains for optimization. , So that the correct pairing ratio of light and heavy chains is increased to over 99%, and the influence of amino acid modification on the expression of mutants is significantly reduced.
  • the first aspect of the present invention is to provide a bispecific antibody comprising: a heavy chain A capable of binding to a specific antigen and a light chain a paired with the heavy chain A, and A heavy chain B that can bind to another specific antigen and a light chain b paired with the heavy chain B; both heavy chain A and heavy chain B have the VH domain of the antibody heavy chain variable region and the antibody heavy chain constant region CH1 Domain, CH2 domain, CH3 domain, light chain a and light chain b all have antibody light chain variable region VL domain and light chain constant region CL domain; one of the VH domain and CH1 domain of heavy chain A Insert a connecting peptide between the VH domain and the CH1 domain of the heavy chain B, and/or insert a connecting peptide between the VL domain and the CL domain of the light chain a, and/or the light chain A connecting peptide is inserted between the VL domain and the CL domain of b; compared with the wild-type human antibody, the heavy chain A and light chain a, heavy chain
  • the connecting peptide inserted between the VH domain and CH1 domain of heavy chain A, the connecting peptide inserted between the VH domain and CH1 domain of heavy chain B, and the VL structure of light chain a may be all the same, or part of the same, or different from each other.
  • the connecting peptide is 1-4 amino acids in length. That is, if it exists, the length of the connecting peptide inserted between the VH domain and CH1 domain of heavy chain A is 1-4 amino acids; if it exists, the length of the connecting peptide inserted between the VH domain and CH1 domain of heavy chain B is It is 1-4 amino acids; if present, the connecting peptide inserted between the VL domain and CL domain of light chain a is 1-4 amino acids in length; if present, it is between the VL and CL domains of light chain b.
  • the connecting peptide inserted in between is 1-4 amino acids in length.
  • the connecting peptide is selected from: G, GG, GS, SG, SS, GGG, GGS, GSG, SGG, GSS, SGS, SSG, SSS, GGGG, GGGS, GGSG, GSGG, SGGG, GGSS, SSGG, GSSG, GSGS, SGSG, SGGS, GSSS, SGSS, SSGS, SSSG, A, AA, AS, SA, SS, AAA, AAS, ASA, SAA, ASS, SAS, SSA, SSS, AAAA, AAAS, AASA, ASAA, SAAA, AASS, SSAA, ASSA, ASAS, SASA, SAAS, ASSS, SASS, SSAS, SSSA, GA, AG, GGA, GAG, AGG, GAA, AGA, AAG, GGGA, GGAG, GAGG, AGGG, GGAA, AAGG, GAAG, GAGA, AGAG, AGGA, GAAA, GAAA, GAGGAA
  • the heavy chain A and heavy chain B, light chain a and light chain b in the bispecific antibody of the present invention contain mutations selected from the following group: (1) The L145 mutation of heavy chain A has positive Charged amino acids, V133 of light chain a is mutated to a negatively charged amino acid, and L145 of heavy chain B is mutated to a negatively charged amino acid, and V133 of light chain b is mutated to a positively charged amino acid; (2) L128 of heavy chain A is mutated to A positively charged amino acid, V133 of light chain a is mutated to a negatively charged amino acid, and L128 of heavy chain B is mutated to a negatively charged amino acid, and V133 of light chain b is mutated to a positively charged amino acid; (3) L145 of heavy chain A Mutation is a positively charged amino acid, V133 of light chain a is mutated to a negatively charged amino acid, and L128 of heavy chain B is mutated to a negatively charged amino acid, and V133 of light chain b is
  • Q39 and L128 of heavy chain A are mutated to positively charged amino acids
  • Q38 and V133 of light chain a are mutated to negatively charged amino acids
  • Q39 and L128 of heavy chain B are mutated to negatively charged Amino acids
  • Q38 and V133 of light chain b are mutated to positively charged amino acids
  • Q39 and L145 of heavy chain A are mutated to positively charged amino acids
  • Q38 and V133 of light chain a are mutated to negatively charged amino acids
  • the heavy chain Q39 and L128 of B are mutated to negatively charged amino acids
  • Q38 and V133 of light chain b are mutated to positively charged amino acids
  • Q39 and L128 of heavy chain A are mutated to positively charged amino acids
  • Q38, V133 of light chain a The mutation is a negatively charged amino acid
  • the Q39 and L145 of the heavy chain B are mutated to negatively charged amino acids
  • the Q38 and V133 of the light chain b are mutated to negatively charged amino
  • the positively charged amino acid refers to K (lysine) or R (arginine), and the negatively charged amino acid refers to D (aspartic acid) or E (glutamic acid).
  • Chain A and heavy chain B, light chain a and light chain b contain mutations selected from the following group: 1) Heavy chain A: L145K or L145R, light chain a: V133D or V133E, and heavy chain B: L145D or L145E, Light chain b: V133K or V133R; 2) Heavy chain A: L128K or L128R, light chain a: V133D or V133E, and heavy chain B: L128D or L128E, light chain b: V133K or V133R; 3) Heavy chain A: L145K Or L145R, light chain a: V133D or V133E, and heavy chain B: L128D or L128E, light chain b: V133K or V133R; 4) heavy chain A: L128K or L128R, light chain
  • Q39R means Gln39 is replaced with arginine (R)
  • Q39K means Gln39 is replaced with lysine (K)
  • Q39E means Gln39 is replaced with glutamic acid (E)
  • Q39D means Gln39 is replaced with arginine (R)
  • Q38K means Gln38 is replaced with lysine (K)
  • Q38E means Gln38 is replaced with glutamic acid (E)
  • Q38D means Gln38 is replaced with aspartic acid (D)
  • L145R means Leucine 145 is replaced with Arginine (R)
  • L145K means Leucine 145 is replaced with Lysine (K)
  • L145E means Leucine 145 is replaced with Glutamate (E)
  • L145D means Leucine 145 is replaced with Aspartic acid (D)
  • L128R means Leucine 128 is replaced with Arginine (R)
  • L128K refers to the replacement of
  • the CH3 domains of heavy chain A and heavy chain B are respectively named as CH3_A domain and CH3_B domain.
  • the CH3_A and CH3_B domains contain The mutations that facilitate the formation of bispecific antibodies are not limited to, for example, WO9627011, CN101198698B, CN102459346B, CN105051069A, US2016177364A1, US2010286374A1, CN106883297A, US20150307628A1, CN104968677A, Nat Biotechnol. 2014 Feb; 32(2):191-8.
  • one of the amino acid mutations at the following positions is involved to facilitate the formation of bispecific antibodies: (a1) CH3_A domain: F405E+K409F+K370D, CH3_B domain: S364R+E357S; ( a2) CH3_A domain: F405E+K409F+K370D+S354C, CH3_B domain: S364R+E357S+Y349C; (a3) CH3_A domain: F405E+K409F+K370D+Y349C, CH3_B domain: S364R+E357S+S354C(b1 ) CH3_A domain: F405E+K409F+K392D, CH3_B domain: D399K; (b2) CH3_A domain: F405E+K409F+K392D+S354C, CH3_B domain: D399K+Y349C; (b3) CH3_
  • Q347R refers to glutamine Gln347 is replaced with arginine (R)
  • Y349C refers to tyrosine Tyr349 is replaced with cysteine (C)
  • S354C refers to serine Ser354 is replaced with cysteine (C)
  • E356K means glutamic acid Glu356 is replaced with lysine (K)
  • E357K means glutamic acid Glu357 is replaced with lysine (K)
  • E357S means glutamic acid Glu357 is replaced with serine (S)
  • K360E means lysine Lys360 is replaced with lysine (K)
  • S364R means serine Ser364 is replaced with arginine (R)
  • S364K means serine Ser364 is replaced with lysine (K)
  • L368D means Leucine Leu368 is replaced with aspartic acid (D)
  • K370D means lysine Lys370 is replaced with
  • the second aspect of the present invention is to provide a composition
  • a composition comprising: (1) the heterodimer according to the first aspect of the present invention, and (2) a pharmaceutically acceptable carrier and/or diluent And/or excipients.
  • the third aspect of the present invention is to provide a polynucleotide comprising: a nucleotide molecule A encoding the heavy chain A of the bispecific antibody of the first aspect of the present invention, encoding the present invention
  • the nucleotide molecule a of the light chain a of the bispecific antibody of the first aspect which encodes the nucleotide molecule B of the heavy chain B of the bispecific antibody of the first aspect of the present invention, encodes the present invention
  • the fourth aspect of the present invention is to provide a vector combination comprising: a recombinant vector selected from the group consisting of a recombinant vector containing a nucleotide molecule A, a recombinant vector containing a nucleotide molecule a, and the nucleotide molecule at the same time Recombination vector of A and nucleotide molecule a, a recombination vector containing said nucleotide molecule B, a recombination vector containing nucleotide molecule b, recombination of said nucleotide molecule B and nucleotide molecule b at the same time
  • Two or more of the vectors, and the combination of vectors contains a nucleotide molecule A, a nucleotide molecule a, a nucleotide molecule B, and a nucleotide molecule b at the same time.
  • the expression vector used in each of the above-mentioned recombinant vectors is a conventional expression vector in the art, which means that it contains appropriate regulatory sequences, such as promoter sequences, terminator sequences, polyadenylation sequences, enhancer sequences, marker genes, and / Or expression vector of sequence and other appropriate sequence.
  • the expression vector may be a virus or a plasmid, such as an appropriate phage or phagemid.
  • Sambrook et al. Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press, 1989.
  • the expression vector of the present invention is preferably pDR1, pcDNA3.1(+), pcDNA3.1/ZEO(+), pDHFR, pTT5, pDHFF, pGM-CSF or pCHO 1.0, more preferably pTT5.
  • the fifth aspect of the present invention is to provide a recombinant host cell containing the vector combination.
  • the original host cell of the recombinant host cell of the present invention can be various conventional host cells in the art, as long as it can make the above-mentioned recombinant vector stably replicate by itself, and the nucleotides carried can be effectively expressed.
  • the original host cell may be a prokaryotic expression cell or a eukaryotic expression cell.
  • the host cell preferably includes: COS, CHO (Chinese Hamster Ovary), NS0, sf9, sf21, DH5 ⁇ , BL21 (DE3 ) Or TG1, more preferably E. coli TG1, BL21 (DE3) cells (expressing single-chain antibodies or Fab antibodies) or CHO-K1 cells (expressing full-length IgG antibodies).
  • the aforementioned expression vector is transformed into a host cell to obtain the preferred recombinant host cell of the present invention.
  • the transformation method is a conventional transformation method in the field, preferably a chemical transformation method, a heat shock method or an
  • the original host cell of the recombinant host cell is preferably a eukaryotic cell, and more preferably a CHO cell or 293E cell.
  • the sixth aspect of the present invention is to provide the bispecific antibody according to the first aspect of the present invention, the composition according to the second aspect of the present invention, the polynucleotide according to the third aspect of the present invention, the present invention Use of the vector combination according to the fourth aspect or the recombinant host cell according to the fifth aspect of the present invention for preparing bispecific antibodies, bispecific fusion proteins and antibody-fusion protein chimeras.
  • the seventh aspect of the present invention provides a method for preparing the bispecific antibody according to the first aspect of the present invention, characterized in that the recombinant host cell according to the fifth aspect of the present invention is used to express the bispecific antibody. Sex antibody.
  • the recombinant host cell contains both the nucleotide molecule A encoding the heavy chain A of the bispecific antibody according to the first aspect of the present invention, and the nucleotide molecule A encoding the bispecific antibody according to the first aspect of the present invention.
  • the nucleotide molecule a of the light chain a of the antibody, the nucleotide molecule B encoding the heavy chain B of the bispecific antibody according to the first aspect of the present invention, and the nucleotide molecule B encoding the bispecific according to the first aspect of the present invention The nucleotide molecule b of the light chain b of the antibody is expressed by the recombinant host cell and recovered to obtain a bispecific antibody.
  • the bispecific antibody can be purified from the recombinant host cell by standard experimental means.
  • protein A can be used for purification. Purification methods include, but are not limited to, chromatographic techniques such as size exclusion, ion exchange, affinity chromatography, and ultrafiltration, or appropriate combinations of the above methods.
  • the molar ratio of nucleotide molecule A, nucleotide molecule a, nucleotide molecule B and nucleotide molecule b in the recombinant host cell is (1-3):(1-3):( 1-3):(1-3), such as 1:1:1:1, 1:1:1.5:1.5, 1:1:2:2, 1:1:2.5:2.5, 1:1:3: 3. 3:3:1:1, 2.5:2.5:1:1, 2:2:1:1, or 1.5:1.5:1:1.
  • the light chain is selected from ⁇ chain or ⁇ chain
  • the constant region is derived from IgG (e.g., IgG1, IgG2, IgG3, IgG4), IgA (e.g., IgA1, IgA2), IgD, IgE or IgM.
  • the CH1 and CL are derived from antibody Fab fragments, preferably from human antibody Fab fragments.
  • the CH1 and CL domains of human antibody Fab fragments are derived from wild-type human antibody Fab fragments.
  • the human antibody Fab fragment of the present invention also includes individual amino acid changes to the wild-type human antibody Fab sequence, for example, including certain amino acid mutations at the glycosylation site, or other nonsense mutations. In addition to the mutations mentioned in the present invention, it may also contain other mutations that do not affect the function of the antibody Fab segment.
  • the CH3 is derived from an antibody Fc fragment, preferably a human antibody Fc fragment.
  • the CH3 domain of a human antibody Fc fragment is derived from a wild-type human antibody Fc fragment.
  • Wild-type human antibody Fc refers to the amino acid sequence that exists in the human population. Of course, there are some subtle differences in Fc fragments among individuals.
  • the human antibody Fc fragment of the present invention also includes individual amino acid changes to the wild-type human antibody Fc sequence, for example, including certain amino acid mutations at the glycosylation site, or other nonsense mutations.
  • the CH3 and CH2 domains may also contain other mutations that do not affect the function of the antibody, especially the Fc segment.
  • the numbering of the amino acid position is determined according to the position of the Kabat EU numbering index.
  • EU index is described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th edition Public Health Service, National Institutes of Health, Bethesda, MD. (1991).
  • amino acid modifications are introduced at specific positions on the antibody light-heavy chain interaction interface, and the heavy chain VH/CH1 connecting peptide and/ Or the connecting peptide between light chain VL/CL has been optimized, so that the correct pairing ratio of light and heavy chains is increased to more than 99%, and the influence of amino acid modification on the expression of mutants is significantly reduced, thereby increasing the yield of antibodies and reducing production costs.
  • Figure 1 shows the results of electrophoresis analysis of light and heavy chain pairing. 4-12% SDS-PAGE protein gel electrophoresis. The lanes from left to right are: protein molecular weight standard, A 2 b, A 6 b, B 12 a, B 16 a.
  • Figure 2 shows the ELISA detection of the binding activity of the EGFR ⁇ HER2 bispecific antibody to the antigen EGFR-ECD-Fc.
  • Figure 3 shows the binding activity of the EGFR ⁇ HER2 bispecific antibody to the antigen HER2-ECD-Fc detected by ELISA.
  • Figure 4 shows the ELISA detection of EGFR ⁇ HER2 bispecific antibody binding activity to the antigens HER2-ECD-Fc and EGFR-ECD-Fc at the same time.
  • Figure 5 shows the binding activity of the EGFR ⁇ cMet bispecific antibody to the antigen EGFR-ECD-Fc detected by ELISA.
  • Figure 6 shows the binding activity of EGFR ⁇ cMet bispecific antibody to antigen cMet-ECD-Fc detected by ELISA.
  • Figure 7 shows the ELISA detection of EGFR ⁇ cMet bispecific antibody binding activity to the antigens cMet-ECD-Fc and EGFR-ECD-Fc at the same time.
  • 293E cells from NRC Biotechnology Research Institute.
  • PBS purchased from Shenggong Biological Engineering (Shanghai) Co., Ltd., catalog number B548117.
  • Citric acid purchased from Sinopharm Chemical Reagent Co., Ltd.
  • Prime star HS DNA polymerase purchased from Takara, product number R010A.
  • Endotoxin-free plasmid large-scale extraction kit purchased from TIANGEN company, item number DP117.
  • HiTrap MabSelectSuRe column purchased from GE Company.
  • AKTA-FPLC fast protein liquid chromatography system purchased from GE Company.
  • Chemidoc MP gel imager purchased from Bio-Rad.
  • G1600AX capillary electrophoresis instrument purchased from Agilent.
  • MicroCal PEAQ-DSC micro calorimeter scanning calorimeter purchased from Malvern Company.
  • Octet molecular interaction system purchased from ForteBio.
  • the four polypeptide chains heavy chain A, light chain a, heavy chain B and light chain b will be randomly paired to produce bispecific antibodies and mismatched antibodies.
  • the light chain a will only specifically pair with the heavy chain A, but will not pair with the heavy chain B.
  • Chain b only specifically pairs with heavy chain B, but not with heavy chain A. Therefore, the antibody light and heavy chains need to be engineered.
  • Table 1 lists the amino acids that interact on the CH1/CL interface of the antibody. By mutating these amino acids, it is possible to promote the correct pairing of the light and heavy chains of the bispecific antibody.
  • the antibody light chain is selected from kappa chain and lambda chain, and the amino acids located on the interaction interface in the light chain constant regions C ⁇ and C ⁇ are highly conserved. Therefore, although all mutations on the antibody light chain in the present invention are completed on the kappa chain, the same applies to the lambda chain.
  • the heavy chain HC (SEQ ID NO: 1) and light chain LC (SEQ ID NO: 2) of the anti-HER2 antibody Trastuzumab were subcloned into the mammalian cell expression vector pTT5 to obtain a recombinant expression vector for mammalian cells to express Trastuzumab.
  • the heavy chain (SEQ ID NO: 3) and light chain (SEQ ID NO: 4) of the anti-EGFR antibody Cetuximab were subcloned into the mammalian cell expression vector pTT5 to obtain a recombinant expression vector for mammalian cells to express Cetuximab.
  • the heavy chain L128 or L145 of Trastuzumab, Cetuximab and anti-IL17Ab and the V133 of the light chain were mutated by overlapping PCR method, and finally the mutant vector for expression in mammalian cells corresponding to the mutants shown in Table 2 was obtained.
  • the expression vector corresponding to the mutation combination of step 1 was transfected into suspension cultured 293E cells with PEI, and the co-transformation ratio of the recombinant expression vector of the heavy chain and the light chain was 1:1. After culturing for 5-6 days, the transient expression culture supernatant was collected, and the antibody expression level was detected by Fortebio using the Fc capture method. The results are shown in Table 2. 1) The wild-type and mutants of all antibodies can be expressed normally, indicating that the introduction of oppositely charged amino acids in the heavy chain (L128 or L145) and light chain V133 does not affect the pairing of the antibody light and heavy chains.
  • CN104968677A and WO2016172485A2 disclose the introduction of oppositely charged amino acids in Q39 of the heavy chain of an antibody and Q38 of the light chain, which is conducive to the correct pairing of the light and heavy chains of the bispecific antibody.
  • the anti-EGFR antibody Panitumumab, the anti-HER2 antibody Trastuzumab, and the anti-cMet antibody Onartuzumab were selected as templates, and the mutation combinations shown in Table 3 were further designed.
  • the heavy chain HC (SEQ ID NO: 9) and the light chain LC (SEQ ID NO: 10) of the above were subcloned into the mammalian cell expression vector pTT5 to obtain a recombinant expression vector for mammalian cell expression.
  • the heavy chain HC and light chain LC encoding genes of Trastuzumab, Panitumumab and Onartuzumab were combined and mutated by overlapping PCR method, and finally recombinant expression vectors for expressing the mutants in mammalian cells were obtained.
  • Example 1 shows that the introduction of mutations in the antibody heavy chain (L128 or L145) and light chain V133 into oppositely charged amino acids does not necessarily affect the expression of the antibody.
  • the present invention further creatively discovered that when the antibody heavy chain (L128 or L145) and light chain V133 mutations are introduced into oppositely charged amino acids resulting in a significant decrease in expression, they can be introduced between antibody VH/CH1 and or between VL/CL
  • the connecting peptide is beneficial to increase the expression level of the mutant.
  • the linker peptide linker1 is inserted before the Ala at position 118 of the EU numbering of the antibody heavy chain
  • the linker peptide linker2 is inserted before the Arg at position 108 of the EU numbering of the antibody light chain.
  • Linker1 and linker2 are linker peptides with a length of 0-4 amino acids. , A connecting peptide sequence of 2 amino acids and 4 amino acids, preferably GG and GGGS. Among them, linker1 is the most obvious linker peptide such as "GG" with 2 amino acids. Accordingly, the mutants shown in Table 4 were constructed. The expression vector corresponding to the mutation combination was transfected into suspension cultured 293E cells with PEI, and the co-transformation ratio of the recombinant expression vector of the heavy chain and the light chain was 1:1.
  • the mutants with the connecting peptide "GG" introduced between the antibody VH/CH1 can all be expressed normally, indicating that the light and heavy chains can be paired correctly. Therefore, the light and heavy chains of combination P2, combination P6, combination P12, and combination P16 were further selected for orthogonal experiment. Specifically, the heavy chain of the combination P2 and the combination P6 is positively charged by mutation, and the light chain is negatively charged, and the heavy chain of the opposite combination P12 and P16 is negatively charged by the mutation, and the light chain is positively charged. Therefore, theoretically, the combination P2, the light chain of the combination P6 and the heavy chain of the combination P12, and the heavy chain of the combination P16 cannot be paired due to the repulsion of the same charge. The light chain of the combination 12, the light chain of the combination P16 and the light chain of the combination P2 and the heavy chain of the combination P6 cannot be paired. There are also repulsions of the same charge that cannot be paired.
  • the heavy chain A of P2 and P6 is named heavy chain A 2 and A 6
  • the light chain is named light chain a
  • the heavy chain B of P12 and P16 is named heavy chain B 12 and B 16
  • the light chain is named light chain b.
  • the anti-EGFR antibody combination P2, P3, P5 and the anti-HER2 antibody combination T4 in Table 4 of Example 2 were randomly selected for light and heavy chain pairing experiments.
  • the light chain of the EGFR antibody and the HER2 antibody and the heavy chain of the EGFR antibody were co-transfected as shown in Table 7 to observe whether the light chain of the HER2 antibody Will interfere with the correct pairing of EGFR antibodies.
  • the EGFR antibody and the light chain of the HER2 antibody were co-transfected with the heavy chain of the HER2 antibody to observe whether the light chain of the EGFR antibody would interfere with the correct pairing of the HER2 antibody.
  • the light chain of HER2 antibody T4 can completely pair with the heavy chain of T4 without interference from the light chain of EGFR antibodies P2, P3, and P5.
  • EGFR antibody P2, P3, and P5 light chains can be correctly paired with EGFR antibody P2, P3, and P5 heavy chains.
  • EGFR antibody P2 is the least affected.
  • P2 antibody The pairing is completely undisturbed.
  • EGFR antibody P2 and HER2 antibody T4 were selected to construct EGFR ⁇ HER2 bispecific antibody.
  • the introduction of the S364R+E357S+Y349C+I253N point mutation in the CH3 domain of the heavy chain of P2 and the F405E+K409F+K370D+S354C point mutation in the CH3 domain of the heavy chain of T4 promoted the preferential formation of heterologous two in the heavy chains of P2 and T4. Aggregate, as shown in Table 8.
  • EGFR-ECD-Fc protein Dilute the recombinant EGFR-ECD-Fc protein with the coating solution to 3 ⁇ g/ml, add 50 ⁇ l/well to the microtiter plate at 4°C overnight. Wash the plate 3 times with PBST, add 200 ⁇ l/well blocking solution, and place it at 37°C for 1 hour, then wash the plate once with PBST for use. Dilute the anti-EGFR ⁇ HER2 bispecific antibody and control antibody (EGFR antibody P2) to 100 ⁇ g/ml with diluent, 4 times the dilution to form 12 concentration gradients (the highest concentration is 100000ng/ml, the lowest concentration is 0.02ng/ml) , Add the blocked ELISA plate successively, 100 ⁇ l/well, and place at 37°C for 1 hour.
  • EGFR antibody P2 anti-EGFR ⁇ HER2 bispecific antibody and control antibody
  • the recombinant HER2-ECD-Fc protein was diluted to 0.4 ⁇ g/ml with coating solution, 50 ⁇ l/well was added to the enzyme-labeled plate, and overnight at 4°C. Wash the plate 3 times with PBST, add 200 ⁇ l/well blocking solution, and place it at 37°C for 1 hour, then wash the plate once with PBST for use.
  • HER2 antibody T4 Dilute the anti-EGFR ⁇ HER2 bispecific antibody and control antibody (HER2 antibody T4) to 100 ⁇ g/ml with diluent, 4 times the dilution to form 12 concentration gradients (the highest concentration is 100000ng/ml, the lowest concentration is 0.02ng/ml) , Add the blocked ELISA plate successively, 100 ⁇ l/well, and place at 37°C for 1 hour. Wash the plate 3 times with PBST, add HRP-labeled mouse anti-human Fab antibody, and place at 37°C for 30 minutes.
  • the recombinant HER2-ECD-Fc protein was diluted to 0.4 ⁇ g/ml with coating solution, 50 ⁇ l/well was added to the enzyme-labeled plate, and overnight at 4°C. Wash the plate 3 times with PBST, add 200 ⁇ l/well blocking solution, and place it at 37°C for 1 hour, then wash the plate once with PBST for use.
  • the anti-EGFR antibody combination P2 and P3 in Example 2 and the anti-cMet antibody combination O2 and O3 were randomly selected for screening.
  • the light chain of the EGFR antibody and the cMet antibody and the heavy chain of the EGFR antibody were co-transfected as shown in Table 9 to observe whether the light chain of the cMet antibody Will interfere with the correct pairing of EGFR antibodies.
  • co-transfect the light chain of EGFR antibody and cMet antibody with the heavy chain of cMet antibody to observe whether the light chain of EGFR antibody interferes with the correct pairing of cMet antibody.
  • the light chains of cMet antibodies O2 and O3 can be completely paired with the heavy chains of O2 and O3, and are not interfered by the expression of EGFR antibodies P2 and P3 light chains.
  • EGFR antibody P2 and P3 light chain can be correctly paired with EGFR antibody P2 and P3 heavy chain. Among them, EGFR antibody P2 is the least affected. When cMet antibody light chain and P2 antibody light chain are transfected in equal proportions, the pairing of P2 antibody is completely different. Be disturbed.
  • EGFR-ECD-Fc protein Dilute the recombinant EGFR-ECD-Fc protein with the coating solution to 3 ⁇ g/ml, add 50 ⁇ l/well to the microtiter plate at 4°C overnight. Wash the plate 3 times with PBST, add 200 ⁇ l/well blocking solution, and place it at 37°C for 1 hour, then wash the plate once with PBST for use.
  • the recombinant cMet-ECD-Fc protein was diluted to 0.4 ⁇ g/ml with coating solution, 50 ⁇ l/well was added to the enzyme-labeled plate, and overnight at 4°C. Wash the plate 3 times with PBST, add 200 ⁇ l/well blocking solution, and place it at 37°C for 1 hour, then wash the plate once with PBST for use.
  • the EC 50 of the anti-EGFR ⁇ cMet bispecific antibody and the positive control cMet antibody O2 bound to cMet-ECD are 0.8635 nM and 0.5524 nM, respectively, and the affinity of the anti-EGFR ⁇ cMet bispecific antibody is the same as that of the cMet single Anti-equivalent.
  • the recombinant cMet-ECD-Fc protein was diluted to 0.4 ⁇ g/ml with coating solution, 50 ⁇ l/well was added to the enzyme-labeled plate, and overnight at 4°C. Wash the plate 3 times with PBST, add 200 ⁇ l/well blocking solution, and place it at 37°C for 1 hour, then wash the plate once with PBST for use.

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Abstract

L'invention concerne un anticorps bispécifique induit par une modification de Fab et son procédé de préparation et son utilisation. En prenant en compte de manière globale diverses interactions entre des acides aminés interfaciaux, telles qu'une interaction électrostatique et une interaction stérique, une modification d'acide aminé est introduite au niveau de positions spécifiques d'une interface d'interaction entre chaîne légère et chaîne lourde d'anticorps. De plus, un peptide de liaison entre VH/CH1 de la chaîne lourde et/ou un peptide de liaison entre VL/CL de la chaîne légère sont optimisés, de sorte que le rapport d'appariement correct des chaînes légère et lourde est accru jusqu'à atteindre au moins 99 %, et l'influence de la modification d'acide aminé sur l'expression du mutant est significativement réduite.
PCT/CN2020/140730 2019-12-31 2020-12-29 Anticorps bispécifique induit par une modification de fab, son procédé de préparation et son utilisation WO2021136249A1 (fr)

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